scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

scholarly journals Autosomal Factor VIII Deficiency in Rabbits: Size Variations of Rabbit Factor VIII.

1977 ◽  
Author(s):  
R.E. Benson ◽  
W.J. Dodds
Keyword(s):  
Molecular Weight ◽  
New Zealand ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Low Molecular Weight ◽  
Antigenic Relationship ◽  
Genetic Studies ◽  
Coagulant Activity ◽  
Autosomal Inheritance

Many rabbits from our Flemish Giant-Chinchilla colony have moderate to severely reduced levels of factor VIII coagulant activity (FVIII-C). Some have shown prolonged bleeding after venipunctures and gastrointestinal and intramuscular hemorrhages. Genetic studies indicate autosomal inheritance. Gel filtration of plasma from these rabbits by the method of Rick et al. (Blood, 49, 209, 1977) at 25°C, pH 6.8 revealed two distinct peaks of FVIII-C; the majority of activity eluting as high molecular weight (HMW) material at the void volume (V°) followed by a much smaller low molecular weight (LMW) peak eluting close to that of fibrinogen. By contrast, filtration of plasma from New Zealand (NZ) rabbits produced threefold greater protein at the V° and equal amounts of HMW and LMW FVIII-C. Increasing the pH to 7.4 had little effect on FVIII-C recovery, although filtration at 4°C virtually abolished the HMW FVIII-C peak of NZ plasma. Rat antiserum (AS) to rabbit HMW FVIII-C, absorbed with precipitate low in FVIII-C, detected precipitating antigen in both HMW and LMW fractions. After absorption with rabbit fibrinogen, the AS no longer detected HMW V° material. The antigenic relationship between HMW and LMW FVIII-C and fibrinogen thus remains unclear. The differences in amount of HMW protein and the ratio of HMW to LMW FVIII-C suggest that in comparison to NZ rabbits our animals have a variant factor VIII molecule as well as low FVIII-C.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Conditions Affecting the Molecular Weight of Factor VIII.

1979 ◽  
Author(s):  
G. Rock ◽  
E. Tackaberry ◽  
D. Palmer
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Calcium Concentration ◽  
Low Molecular Weight ◽  
Biochemical Purification ◽  
Molecular Weights ◽  
Will Self ◽  
And Storage ◽  
Molecular Weight Form

By purifying Factor VIII while maintaining physiological concentrations of calcium we have recently demonstrated that about 50% of the procoagulant activity is in a very low molecular weight (VLHW) form not associated with the carrier (VIII: RAG). The remainder is carrier associated and elutes at Vo as a high molecular weight (HMW) compound upon Sepharose 6B chromatography. Reduction of the calcium concentration by increasing the amount of citrate added to heparin results in decreasing the ratio of VLMW:HMW from 1:1 in pure heparin to 1:5 in pure citrate. If citrate is replaced with the more strongly chelating EDTA no VLMW is detectable in the plasma. It has also been found that most of the biochemical purification techniques which have been previously used to prepare Factor VIII for study actually result in the aggregation of this VLMW with the carrier to produce the high molecular weight form. This includes: cryoprecipitation, precipitation by polyethylene glycol and storage -80°C. As well, the VLMW material will self-associate upon freezing to produce an aggregate with a molecular weight of 106. However, this material does not cross-react with rabbit antibody directed against VIII: RAG. The data indicate that many of the previously reported biochemical characteristics, including molecular weights, actually describe species which are artifacts of the isolation process rather than those of the physiologically occuring Factor VIII.

Characterization Of Soluble DES-A And DES-AB Fibrin In Human Plasma At 37°C By Agarose-Gel Filtration

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J-C Bernhard ◽  
I Mahn
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Agarose Gel ◽  
High Molecular Weight Material ◽  
Different Temperatures ◽  
Lower Temperature

In two consecutive steps, thrombin cleaves the fibrinopeptides A and B from fibrinogen producing des-A fibrin and des-AB fibrin. Labeled des-A fibrin was prepared by batroxobin and labeled des-AB fibrin by clotting of 125I-fibrinogen with thrombin. Fibrin solubilized in buffered urea was mixed with plasma containing 131I-fibrinogen (fibrin:fibrinogen ratio = 1:20). These fibrinfibrinogen mixtures were applied to sepharose CL- 6B columns eq ui librated with buffered plasma (0.0025 M EDTA, 0.1 M NaCl, 0.05 M tris, 0.005 M EACA, 2 AT U hirudin/ml, 500 KIU a protinin/ml, 0.003 M NaN3, pH 7.4). Plasma was used as an equilibration and elution medium to prevent precipitation of fibrin in the columns. At 20°C, labeled des-A fibrin as well as des-AB fibrin were eluted in the void volume as high-molecular weight aggregates (peak A) and separated from m onomeric labeled fibrinogen (peak B). At 37°C, however, des- A fibrin was eluted at the same position as monomeric fibrinogen (peak B), whereas des-AB fibrin was eluted in the void volume as at 20°C. Rechromatography of isolated fractions of peak A and peak B at different temperatures showed that monomeric fibrin isolated at 37°C formed high molecular weight material at 20°C, and high-molecular weight fibrin isolated at 20°C dissociated at 37 ° C. The results suggest that des-A fibrin solubilized in plasma in the absence of calcium ismonomeric at 37°C but forms high-molecular weight aggregates at lower temperature, whereas des-AB fibrin is oligomeric at 20°C as well as at 37°C.

Comparison Of Naturally Occurring And CaCl2-Separated Canine Factor VIII-Coagulants Free Of FACTOR VIII-Related Antigen

1981 ◽  
Author(s):  
R E Benson ◽  
W J Dodds
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Room Temperature ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Lower Molecular Weight ◽  
Elution Volume ◽  
Stable Factor ◽  
Naturally Occurring ◽  
Similar Combination

Previous studies demonstrated that plasma from dogs homozygous for von Willebrand’s disease (VWD) without detectable factor VUI-related antigen (VIII:RAg) contained moderate levels of a stable factor VIH-coagulant (VIII:C), which had lower apparent molecular weight and combined with the VIII:RAg of canine hemophilic plasma. We have now compared this VWD-VIII:C to the CaCl2-separated form of VIII:C prepared from normal canine factor VIII. The VWD plasma was fractionated at room temperature by 6% agarose gel filtration in 0.15M NaCl and 0.24M CaCl2 buffers, pH 7.25; the VIII:C eluted in each buffer with a relative elution volume (Ve/Vo) of 1.4. Isolated lower molecular weight VIII:C stabilized with albumin (5 mg/ml) and dialysed free of Ca++ was prepared from normal purified canine factor VIII by elution in 0.24M CaCl2 buffer. This CaCl2~separated VIII:C was rechromatographed in both buffers as above and had a Ve/Vo of 1.9. When the VWD and CaCl2 forms of VIII:C were each combined with canine hemophilic plasma and gel filtered in 0.15M NaCl buffer, the VIII:C’s now appeared at the void volume. These mixtures with 1/10 volume 2.4M CaCl2 added before or after combination were also analyzed in 0.24M CaCl2 buffer. The CaCl2-VIII:C eluted with a Ve/Vo of 1.9 regardless of the order of 2.4M CaCl2 addition, but the VWD-VIII:C eluted with Ve/Vo’s of 1.4 when CaCl2 was added before mixing and 1.9 when added afterwards.Thus, when mixed with hemophilic plasmas both the CaCl2 and VWD forms of VIII:C exhibited similar combination and separation characteristics. However, the finding that the CaCl2-VIII:C is of apparent lower molecular weight than the VWD form suggests that the CaCl2-separated VIII:C is different from the naturally occurring form of VWD (VIII: RAg-free) VIII:C.

scholarly journals Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

1979 ◽  
Vol 179 (3) ◽  
pp. 515-523 ◽  
Author(s):  
Thomas E. Knauer
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Supernatant Fraction ◽  
Ph Optimum ◽  
Factors Affecting ◽  
Chain Acyl ◽  
Coa Thioesters ◽  
Molecular Weight Form

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10μm-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl2, and 5,5′-dithiobis-(2-nitrobenzoic acid), and exhibit the same Km (1.8μm) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0°C), but there is no change in the Km with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10μm-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

Bovine Factor VIII: Purification of the Procoagulant Protein

1979 ◽  
Author(s):  
G. A. Vehar ◽  
E. W. Davie
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Factor Xa ◽  
Apparent Molecular Weight ◽  
Von Willebrand Disease ◽  
Factor X ◽  
Coagulant Activity ◽  
Von Willebrand

Factor VIII has been purified from bovine plasma approximately 500,000 fold with an overall yield of about 5%. Purification steps included affinity chromatography as well as DEAE-Sephadex and sulfate-Sepharose chromatography and gel filtration on Sephadex G-200. The final product readily corrected the clotting of plasma from patients with hemophilia A or severe von Willebrand disease. The purified preparation had no von Wil1ebrand factor activity, as measured by aggregation of platelet - rich human plasma. The coagulant activity was inhibited by antibodies prepared against a purified von Willebrand/factor VIII preparation and was required for the conversion of factor X to factor Xa in the presence of factor IXa, calcium, and phospholipid. The highly purified preparation appeared as a triplet on urea-SDS polyacrylamide gels with an apparent molecular weight of about 70,000. Preincubation of factor VIII with thrombin or factor Xa resulted in (20-50 fold increase in the coagulant activity, and this increase correlated with a reduction in the molecular weight of the protein as determined by SDS polyacrylamide gel electrophoresis. Activated factor VIII was inhibited by preincubation with DFP, in contrast to the precursor form. These data indicate that factor VIII is a trace protein in plasma in a precursor form and is converted to an activated form during the coagulation process. (Supported by NIH Grant HL 16919 and the National Hemophilia Foundation.)

Inhibitors of Urokinase-Induced Fibrinolysis in Pregnancy

1979 ◽  
Author(s):  
J. E. Duthie ◽  
D. M. Campbell ◽  
D. Ogston
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Inhibitory Activity ◽  
Substantial Reduction ◽  
Gel Filtration ◽  
Late Pregnancy ◽  
Lower Molecular Weight ◽  
Α2 Macroglobulin ◽  
Molecular Weight Fractions ◽  
In Pregnancy

Pregnancy plasma possesses inhibitory activity against urokinase measured on unheated fibrin plates. Antiurokinase activity in late pregnancy plasma subjected to gel filtration on Sephadex G-200 eluted with the high molecular weight proteins including α2-macroglobulin and, in greater quantity, with albumin. In all non-pregnancy plasmas the high molecular weight inhibitor activity was present in equivalent quantities; the lower molecular weight inhibitor was found in small amounts in only a proportion of plasmas. The anti-urokinase activity of pregnancy plasma could be separated from α1-antitrypsin and α2-antiplasmin by chromatography on DEAE-Sephadex. Within 1 hour of parturition plasma fibrinolytic activity increased and there was substantial reduction in the anti-urokinase activity of the lower molecular weight fractions; no change was seen in the high molecular weight inhibitory activity. It is concluded that anti-urokinase activity in pregnancy plasma resides in a protein distinct from established protease inhibitors; a placental source is postulated.

scholarly journals Presence of a high-molecular-weight form of catalase in enzyme purified from mouse liver

1977 ◽  
Vol 163 (3) ◽  
pp. 449-453 ◽  
Author(s):  
M B Baird ◽  
H R Massie ◽  
L S Birnbaum
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
High Molecular Weight Form ◽  
High Molecular Weight Material ◽  
Molecular Weight Form

Ultracentrifugation studies of purified mouse hepatic catalase revealed that 5-7% of the total material consists of a form with a higher molecular weight than the bulk of the catalase. The two components were separated by sucrose-gradient centrifugation. Polyacrylamide-gel electrophoresis (in borate buffer) demonstrated that high-molecular-weight catalase is enriched in a more slowly migrating component, and sodium dodecyl sulphate/polyacrylamide gel-electrophoresis demonstrated that the molecular weight of the subunits of the high-molecular-weight material is identical with that of the subunits of the major form. These results suggest that high-molecular-weight catalase consists of subunits that are not markedly distinct from those present in the normal catalase tetramer.

Immunologic evidence that the properties of human antihemophilic factor (factor VIII) are attributes of a single molecular species

Blood ◽  
1976 ◽  
Vol 47 (4) ◽  
pp. 657-667 ◽  
Author(s):  
OD Ratnoff ◽  
CC Slover ◽  
MC Poon
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Molecular Species ◽  
Gel Filtration ◽  
Antigenic Sites ◽  
Heterologous Antiserum ◽  
Aggregation Of Platelets ◽  
Antihemophilic Factor

Abstract Preparations of human plasma rich in antihemophilic factor (AHF, factor VIII) correct the coagulative defect of classic hemophilic plasma, form precipitates with specific heterologous antiserum, and support aggregation of platelets by ristacetin and retention of platelets by columns of glass beads. Whether these various properties can all be attributed to a single molecular species is disputed. Antiserums were prepared in rabbits to partially purified AHF and to high molecular weight (MW) and low MW fragments separated by gel filtration through columns of agarose in the presence of 0.25 M calcium chloride. Antiserums to AHF and to its high or low MW fragments all inactivated procoagulant AHF in plasma or in preparations of AHF. In contrast, antiserums to AHF and its low MW fragment inactivated procoagulant AHF in the low MW fragment, while that against the high MW fragment lacked this property. Thus, the low MW fragment appeared to have some antigenic sites not present or accessible to the antiserum against the high MW fragment. In agreement with this, the low MW fragment did not block antiserum against the high MW fragment as tested by the capacity of this antiserum to inactivate functional AHF in plasma. These immunologic studies support the view that the various properties of preparations of human AHF are attributes of a single molecular species.

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